1. Field of the Invention
The present invention relates to a method of quantitative microorganism assay for a vitamin B.sub.12 (hereafter simply referred to as B.sub.12)-containing substance and a reagent used for the method of quantitative assay. More particularly, the present invention relates to a method of quantitative assay of B.sub.12 in a B.sub.12 -containing substance using marine methanol-utilizing bacteria requiring B.sub.12 for growth and capable of assimilating methanol quickly in a simple manner and a reagent for quantitative assay of the B.sub.12 -containing substance comprising a composition of dry viable bacteria for inoculation of marine methanol-utilizing bacteria having B.sub.12 auxotrophy used for the quantitative assay and a liquid medium containing methanol and also containing pyrroloquinoline-quinone and a polyoxyethylene sorbitan fatty acid ester and a B.sub.12 standard solution.
2. Brief Description of the Prior Art
Physicochemical methods and microbiological methods have been hitherto known as quantitative assay for B.sub.12 -containing substances, i.e., substances containing vitamin B.sub.12. Among the microbiological methods, a method of quantitatively determining B.sub.12 in B.sub.12 -containing substances using marine methanol-utilizing bacteria requiring B.sub.12 for growth is known to be excellent (Published Unexamined Japanese Patent Application No. 19022/1980).
When an attempt is made to quantitatively determine B.sub.12 in B.sub.12 -containing substances, however, this method is disadvantageous in that bacteria do not necessarily have stable growth and the B.sub.12 cannot be quantitatively determined quickly with accuracy.
Problems to be solved by the present invention are in the development of an improved method for quantitatively assaying a B.sub.12 -containing substance that eliminates the defects possessed by conventional assay methods of B.sub.12 -containing substances using marine methanol-utilizing bacteria having B.sub.12 auxotrophy, namely, failing to quantitatively determine the B.sub.12 -containing substance stably and quickly with good accuracy, and reagents for quantitative assay for B.sub.12.
As a result of various investigations to solve the foregoing problems, the present inventors have found that by supplementing pyrroloquinoline-quinone and a polyoxyethylene sorbitan fatty acid ester to a medium for quantitative assay using marine methanol-utilizing bacteria having B.sub.12 auxotrophy, the B.sub.12 in a B.sub.12 -containing substance can be quantitatively determined stably with accuracy. The present invention has been accomplished based on this finding.